Small molecule inhibitors selective for polo-like kinase proteins

ABSTRACT

Disclosed are small molecule PLK inhibitors that can target the polo box domain (PBD). Inhibitors can have an atomic mass of about 1000 Da or less and a general structure ofFor instance, the inhibitors can include an alkyl benzamido benzoic acid core structure.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 15/479,373, having a filing date of Apr. 5, 2017,which claims filing benefit of U.S. Provisional Patent Application Ser.No. 62/318,439, having a filing date of Apr. 5, 2016, both of which areincorporated herein by reference for all purposes.

SEQUENCE LISTING

This application contains a Sequence Listing, which is filedconcurrently herewith in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 20, 2017, isnamed USC-471_Sequence_List.txt and is 923 bytes in size.

BACKGROUND

The family of polo-like kinase (PLK) proteins are central players inregulating entry into and progression through mitosis. The four knownhuman PLKs have non-redundant and non-overlapping functions. Asignificant body of literature has validated PLKs as anti-tumor drugtargets. For instance, profound anti-proliferative activity can beachieved through selective inhibition of PLK1 functions. Over-expressionof PLK1 is frequently observed in various cancers and PLK1 expression isa prognostic indicator for outcome of patients suffering from varioustumors. For example, more than half of prostate cancers over expressPLK1 and this expression is positively correlated with tumor grade.

The therapeutic rationale for PLK inhibition has been validated throughstudies with PLK1-specific antisense oligonucleotides and has been shownto profoundly inhibit cancer cell growth in vitro and in vivo whilehaving little effect on the proliferation of normal cells. Severalstudies also suggest that lethality of PLK1 against cells with mutationsin p53, KRAS, and PTEN can be exploited to selectively kill tumor cells,and this research provides additional target validation. Numerousinhibitors that target ATP binding to PLKs are being clinicallyevaluated. Published results suggest acceptable toxicity profiles, thuswarranting further investigation and the phase II trials underway.Through such research, a small molecule PLK1 inhibitor BI6727(volasertib) was granted breakthrough status after significant benefitwas observed in treating Acute Myeloid Leukemia.

Unfortunately, despite clinical progress, there are significantdrawbacks for PLK inhibitors that target the ATP cleft. For instance,all ATP competitive protein kinase inhibitors suffer from the well-knownconcerns of relative non-specificity in the human kinome and of havingto compete against high intracellular ATP concentrations. Moreover, evenif ATP cleft-targeted PLK inhibitors progress fully through clinicaltrials, recent experience with other kinase inhibitors suggests thattumor resistance will be a problem in the future. A prime example is thefirst clinically validated kinase inhibitor, imatinib. While this drugwas a remarkable breakthrough for chronic myelogenous leukemia, pointmutations in the active site of BCR-ABL were observed soon afterapproval, leading to clinical resistance. It took over a decade ofintense research to develop ponatinib, the first FDA-approved drugeffective against the T315I mutant kinase. The probability of a similarscenario with PLK1 inhibition was underscored with a recent publicationshowing that a single point mutation in PLK1 (C67V) confers substantialresistance to BI2536 and several other structurally unrelated andclinically evaluated ATP inhibitors while also not affecting kinaseactivity. In fact, such selective pressure might partially beresponsible for the lack of clinical activity of BI2536 as a monotherapydespite dramatic preclinical anti-tumor activity.

Another drawback of ATP competitive compounds arises from the knowledgethat three of the mammalian PLKs are inhibited equipotently by BI2536.In particular, while this compound is more selective for PLK1, it stillhas nanomolar activity against PLK3. Studies have shown PLK3 to be atumor suppressor with activities that oppose those of PLK1, and severalstudies have provided evidence of opposing functions of PLK1 and PLK3.Thus, anti-tumor activity of ATP inhibitors against PLK1 may bediminished by concomitant inhibition of PLK3.

Accordingly, alternative approaches and effective small molecule PLKinhibitors are needed in the art. Moreover, highly selective PLKinhibitors, and in particular, highly selective PLK1 inhibitors, wouldbe of great benefit in the art.

SUMMARY

According to one embodiment, disclosed is a non-peptidic small moleculePLK inhibitor having the general structure of:

wherein

R₁ is an alkyl, —O-alkyl, —S-alkyl, or —NH-alkyl, and optionallyincludes a terminal aryl;

R₈ and R₉ are independently H, alkyl, OH, NH₂, SH, —O-alkyl, —NH-alkyl,—S-alkyl, —CONH-alkyl, halogen, or —CN;

X is an amide, —C1 to C4 alkyl-NH—, —NH—, —SNH—, —SONH—, or —SO₂N—;

R₇ is selected from the following structures:

in which R₂ through R₆ are independently selected from hydrogen,carboxyl, alkyl, alkyl ester, hydroxyl, methoxy, halogen, sulfonamide,phosphate, nitro, methylamine, or boronic acid, and

R₁₀ is C or N, and R₃ is relevant only when R₁₀ is C;

According to another embodiment, small molecule inhibitors exhibitingselectivity for PLK1 or PLK3 are described. For instance, a PLK1selective small molecule inhibitor is disclosed having the generalstructure as described above in which R₁ is a C3 to C8 alkyl, R₈ and R₉are hydrogen, R₇ is a), R₂ is carboxyl, R₃ through R₆ are independentlyselected from hydrogen, hydroxyl, methoxy, halogen, nitro, or alkyl, R₁₀is C, and X is an amide linkage.

In one embodiment, a PLK3 selective small molecule inhibitor isdescribed. For example a PLK3 selective small molecule inhibitor canhave the general structure as described above in which R₁ is a C6 to C12alkyl, R₈ and R₉ are hydrogen, R₇ is a), R₂ is hydrogen or carboxyl, R₃through R₆ are independently selected from hydrogen, carboxyl, methyl,or hydroxyl, R₁₀ is C, and X is an amide linkage.

In another embodiment, the PLK selective inhibitor can have the abovestructure wherein R₁ is alkyl, R₈ and R₉ are hydrogen, X is an amidelinkage, and R₇ is one of b) through g).

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood with reference to thedrawings, in which:

FIG. 1 is a table of exemplary inhibitors as described herein (SCCP IDnumbers provided on FIG. 1 are utilized throughout this disclosure).

FIG. 2 illustrates the binding mode of inhibitor SCCP ID No. 5905 withthe PBD of PLK1.

FIG. 3 illustrates the dose dependent G2/M arrest induced by two of theinhibitors (SCCP ID Nos. 5915 and 6037) of Table 1.

FIG. 4 presents a scheme for development of the small moleculeinhibitors.

FIG. 5 presents exemplary synthesis schemes for formation of theinhibitors.

FIG. 6 graphically illustrates the resistance of retinal pigmentepithelial (RPE) cells to BI-2536, an ATP-competitive inhibitor.

FIG. 7 graphically illustrates the sensitivity of RPE cells to aninhibitor (SCCP ID No. 5912) as disclosed herein.

FIG. 8 graphically illustrates the sensitivity of RPE cells to anotherinhibitor (SCCP ID No. 5932) as disclosed herein.

FIG. 9 provides cell cycle analysis of several disclosed PLK inhibitorsin prostate cancer (PC3) cells.

FIG. 10 provides cell cycle analysis of one small molecule PLK inhibitor(SCCP ID No. 5912) in prostate cancer (PC3) cells.

FIG. 11 provides cell cycle analysis of another small molecule PLKinhibitor (SCCP ID No. 6037) in prostate cancer (PC3) cells.

FIG. 12 provides cell viability analysis for a small molecule inhibitor(SCCP ID No. 6037) as described herein.

FIG. 13 provides cell viability analysis of another small moleculeinhibitor (SCCP ID No. 5914) as described herein at variousconcentrations for both the inhibitor and the cells.

FIG. 14 provides cell viability analysis of another small moleculeinhibitor (SCCP ID No. 5915) as described herein.

DETAILED DESCRIPTION

The following description and other modifications and variations to thepresently disclosed subject matter may be practiced by those of ordinaryskill in the art, without departing from the spirit and scope of thepresent disclosure. In addition, it should be understood that aspects ofthe various embodiments may be interchanged both in whole and in part.Furthermore, those of ordinary skill in the art will appreciate that thefollowing description is by way of example only and is not intended tolimit the subject matter.

The present disclosure is generally directed to small molecule PLKinhibitors that can target the polo box domain (PBD). All PLKs containan N-terminal Ser/Thr kinase catalytic domain and a C-terminal regionthat includes the PBD. In the absence of a bound substrate, the PBDinhibits the basal activity of the kinase domain.Phosphorylation-dependent binding of the PBD to its ligands releases thekinase domain, while simultaneously localizing polo-like kinases tospecific subcellular structures. Thus, the PBD is critical for PLKsubcellular localization and substrate recognition prior tophosphorylation.

PLK1 localizes to centrosomes and kinetochores in prophase and to thespindle midzone later in mitosis, which depends on the PBD but not onits kinase activity. For instance, a PBD fragment fused with amembrane-permeable delivery peptide can cause mitotic arrest and celldeath in tumor cells. Also, inducible expression of the PLK1 PBD domainfragment in PC-3 prostate cancer cells has been shown to result insignificant growth inhibition, validating the concept that interferingwith the PBD suppresses the proliferative effects of PLK1.Crystallography has revealed the molecular basis for PLK1 localizationthrough the PBD and peptide inhibitors have been identified. The lack ofsuccess of HTS approaches confirms that alternative approaches to PBDinhibitor development are desirable.

The PLK inhibitors disclosed herein are small molecule inhibitors thatcan target the PBD. As utilized herein, the term “small molecule” refersto a non-peptidic compound that is generally about 1000 Daltons or less(i.e., atomic mass units, one Dalton being equivalent to 1/12 the massof a ¹²C isotope). In other embodiments, the small molecule inhibitormay be about 500 Daltons or less, about 400 Daltons or less, or about300 Daltons or less.

Beneficially, the small molecule PBD-targeted inhibitors can retainactivity exhibited by peptidic inhibitors, e.g., antitumor activityagainst cancer cells, and, in one particular embodiment, can exhibitactivity against cancer cells that can acquire resistance to ATP-basedinhibitors. As such, in one embodiment, disclosed inhibitors can be usedin combination with ATP-based inhibitors as a synergistic means of PLK1targeting in the clinic. Moreover, by targeting non-catalytic functions,PLK can be less likely to obtain resistance to the inhibitors.

The non-peptidic inhibitors have been developed with activity andcellular phenotypes consistent with target engagement of PLK1, and, inone embodiment, can induce apoptosis. As such, the inhibitors can beuseful as mechanistic probes to characterize cellular defects ofblocking PLK1 independent of catalytic activity, and can be used incombination with catalytic PLK1 inhibitors as a dual approach toattacking PLK1, analogous to many successful clinical precedents (e.g.,Bactrim (antibiotic), Combivir® (antiviral), pertuzumab and trastuzumabused in HER2 breast cancer).

Disclosed PBD-targeted inhibitors that are specific for PLK1 are alsomuch less likely to affect the activity of the PLK3 tumor suppressor, ascertain of the PLK1 PBD domain inhibitors have minimal activity againstPLK3, as discussed further herein.

The small molecule inhibitors can exhibit comparable affinity topeptidic PBD inhibitors and can possess anti-proliferative phenotypes incells consistent with the observed decrease in PLK1 centrosomallocalization. The inhibitors can demonstrate evidence of enhanced PLK1inhibition in cells relative to peptides and can induce monopolar andmultipolar spindles, in contrast to previously reported small moleculePBD inhibitors that display phenotypes only partially representative ofPLK1 knockdown. The inhibitors can function as isotype,kinase-selective, non-ATP competitive inhibitors and can be utilized asPLK1 selective anti-tumor therapeutics.

The small molecule inhibitors described herein include an alkylbenzamido benzoic acid core structure that has been utilized to buildthe PBD inhibitors with high potency and selectivity. As utilizedherein, unless otherwise noted, the term “alkyl” refers to anystraight-chain or branched, substituted or unsubstituted C1 to C20 alkylgroup.

In one embodiment, a non-peptidic small molecule PLK inhibitor can havethe general structure of:

wherein

R₁ comprises an alkyl, —O-alkyl, —S-alkyl (e.g., —S—CH₃), or —NH-alkyl,and optionally includes a terminal aryl;

R₈ and R₉ are independently hydrogen, alkyl, OH, NH₂, SH, —O-alkyl,—NH-alkyl, —S-alkyl, —CONH-alkyl, halogen, or —CN;

X is an amide (i.e., either —C═O—NH— or —NH—C═O—), —C1 to C4 alkyl-NH—,—NH—, —SNH—, —SONH—, or —SO₂N—;

R₇ is selected from the following structures:

in which R₂ through R₆ are independently selected from hydrogen,carboxyl, alkyl, alkyl ester, hydroxyl, methoxy, halogen, sulfonamide,phosphate, nitro, methylamine, or boronic acid, and

R₁₀ is C or N, and R₃ is relevant only when R₁₀ is C;

For example, in one embodiment, the small molecule inhibitors can havethe general structure of:

wherein

-   -   R₁ comprises an alkyl, —O-alkyl, —S-alkyl, or —NH-alkyl, and        optionally includes a terminal aryl;    -   R₂ through R₆ are independently selected from hydrogen,        carboxyl, alkyl, alkyl ester, hydroxyl, methoxy, halogen,        sulfonamide, phosphate, nitro, methylamine or boronic acid; and    -   X is an amide, —C1 to C4 alkyl-NH—, —NH—, —SNH—, —SONH—, or        —SO₂N—.

FIG. 1 presents the structure, structure activity relationship (SAR) andinteraction data of several representative small molecule inhibitors ofthis embodiment. A clear SAR for substituents at the 4 position ofincreasing alkyl chain length has been established and provides a solidbasis and rationale for improvements in potency.

In another embodiment, the small molecule inhibitors can have thegeneral structure of:

wherein

-   -   R₁ comprises an alkyl, —O-alkyl, —S-alkyl, or —NH-alkyl, and        optionally includes a terminal aryl;    -   R₇ is selected from:

Table 1, below, below presents the structure of several representativePLK inhibitors of this embodiment.

TABLE 1 SCCP PLK1 PBD ID No. R₁ X R₇ IC₅₀ (μM) 5992 octyl amide b),R₁₁═N ND 5904 hexyl amide b), R₁₁═N >600 5913 octyl amide d), R₁₁═CH 1055975 octyl amide f) >200 5976 octyl amide g) 343-450 5997 octyl amidec), R₁₁═CH ND

FIG. 2 schematically illustrates the binding mode of one representativeinhibitor (SCCP ID No. 5905 of FIG. 1 ) with the PBD of PLK1. Theinteraction of the carboxylate at R₂ with the phosphate binding site isshown by the dashed line. The unoccupied regions that can be exploitedto improve the inhibitor affinity further are indicated in FIG. 2 by theS1-S4 labels. The SAR and predicted interactions of the hydrophobic slotin the PBD (FIG. 2 ) indicates that linear alkyl groups can result in anenhancement in potency. As indicated in FIG. 1 , synthesis of the 4substituted analogs, including n-propyl, butyl, hexyl, and octyl groups,led to improved potencies.

The alkyl benzamido benzoic acid core provides a scaffold that exhibitsexcellent drug development potential. The small molecule inhibitors haverelatively low molecular weight, possess high ligand efficiency, have alow polar surface area (e.g., about 200 Å² or less, about 100 Å² orless, or about 75 Å² or less in some embodiments, e.g., 66 Å² for SCCPID No. 5912) and can have a C log P of about 5. In addition, they arerelatively simple to synthesize by standard chemical practice (examplesof which are described further herein and have considerable scope forhigh activity through expansion of the molecules into unoccupied regionsof the PBD groove (FIG. 2 )).

The small molecule inhibitors can be designed to exhibit PLK selectivityand/or predetermined activity. By way of example, SCCP ID No. 5881 (FIG.1 ) has low micromolar activity in the binding assay and possesses a4-hexylbenzamide and 2-aminobenzoic acid bonded with an amide linkage.Modeling with the PBD of PLK1 has illustrated that the ortho-carboxylicacid is a bioisostere for the phosphate group in the peptide context. Anumber of variants of SCCP ID No. 5881 have been synthesized and the SARexamined, with results provided in FIG. 1 . As shown, movement of thecarboxylate to the R₃ position substantially decreased the activity(SCCP ID No. 5903).

SAR can be predetermined in one embodiment by varying the length of thealkyl chain at the R₁ location. For example, decreasing the length canreduce binding potency whereas increasing it can increase bindingpotency. For example, increasing the length of the alkyl chain to 8carbons (e.g., SCCP ID No. 5912) and to 12 carbons (e.g., SCCP ID No.6037) resulted in notable increases in binding activity relative to SCCPID No. 5881.

The inhibitors can include a halogen moiety at one or more of the R₂through R₆ locales. For example, an R₆-fluoro derivative was synthesized(SCCP ID No. 5932) and shown to have similar activity relative to SCCPID No. 5912.

The inhibitors can include an alkyl moiety at one or more of the R₂through R₆ locales, which can be utilized to provide predeterminedactivity and/or selectivity. For example, addition of a methyl group atthe R₅ position of the benzoic acid ring (SCCP ID No. 5915) resulted inan almost 5-fold increase in activity for PLK1 as compared to SCCP IDNo. 5912, whereas a methyl group at R₄ (SCCP ID No. 5937) provided lowerPLK1 activity.

In some embodiments, the disclosed inhibitors can discriminate betweenPLK family members. Beneficially, the inhibitors can replicate a PLK1phenotype in contrast to the partial phenotype obtained with PBDdominant negative and small molecule inhibitors utilized in the past.Thus, it is believed that inhibitors formed, as described herein, caninhibit both subcellular localization and substrate phosphorylation.

As illustrated in FIG. 1 , the small molecule inhibitors can be designedto exhibit preferential specificity between PLK1 and PLK3. For example,structures that include a simple benzoic acid moiety can possess lessspecificity for PLK1 versus PLK3. However, other compounds, such as SCCPID No. 5915, with a methyl group at R₄, exhibit a high selectivity forPLK1. SCCP ID No. 5912, with an R₁ octyl substituent is relativelyequipotent for PLK1 and PLK3 PBD binding, whereas SCCP ID No. 6037 withan R₁ dodecyl group shows selectivity for PLK3.

Without wishing to be bound to any particular theory, it is believedthat interactions with the unoccupied hydrophobic pocket (FIG. 2 , S3),through derivatization at the R₄ and R₅ positions of the inhibitorstructure, can lead to the high PLK1 vs. PLK3 selectivity. By way ofexample, a small molecule inhibitor exhibiting high PLK 1 selectivitycan have the following general structure:

wherein

-   -   R₁ comprises a C3 to C8 alkyl or —O—C3 to C8 alkyl and        optionally includes a terminal aryl;    -   R₂ is carboxyl;    -   R₃ through R₆ are independently selected from hydrogen,        hydroxyl, methoxy, halogen, or alkyl;    -   Y is a C═O (i.e., an amide linkage).

In one particular embodiment, a PLK1 selective small molecule inhibitorcan have the above structure in which R₃ and R₆ are hydrogen and R₄ andR₅ are independently selected from hydrogen, hydroxyl, methoxy, halogenor alkyl, wherein at least one of R₄ and R₅ is not hydrogen.

In another embodiment, the small molecule inhibitor can be selective forPLK3. For example, a PLK3 selective small molecule inhibitor can havethe general structure of:

wherein

-   -   R₁ comprises a C6 to C12 alkyl;    -   R₂ is hydrogen or carboxyl;    -   R₃ through R₆ are independently selected from hydrogen,        carboxyl, methyl, or hydroxyl; and    -   Y is a C═O (i.e., an amide linkage).

In one embodiment, a PLK3 selective inhibitor can have the abovestructure in which R₃ through R₆ are hydrogen and optionally, the R₁group can be a C12 alkyl chain (e.g., SCCP ID No. 6037).

PLK specificity can be useful in therapeutic applications, as well as inresearch-oriented application. For example, a differential cell cycleeffect has been observed dependent on the selectivity (PLK1 vs. PLK3) ofthe inhibitor, and this effect has moreover been observed to be dosedependent. Untreated cells can progress through one cell cycle and begintransit through a second cycle (47% G1 and 37% S phase by DAPI, 62%positive for BrdU labeling, demonstrating a majority of the cells areactively replicating early in S phase). As shown in FIG. 3 , cellstreated with SCCP ID No. 6037 (PLK3 selective) at 5 μM revealed aprofound block in G1 after progressing through one mitosis (FIG. 3 ,2^(nd) panel: 77% G1 and 15% S by DAPI, only 27% BrdU+). Release into ahigher dose of 20 μM, SCCP ID No. 6037 resulted in a significant blockprior to mitosis (FIG. 3 3^(rd) panel; 28% G2/M by DAPI, compared to 13%for vehicle and 7% for 5 μM, SCCP ID No. 6037). In contrast, release ofPC3 cells in SCCP ID No. 5915 (PLK1 selective), resulted in a clear G2/Maccumulation (FIG. 3 , 4^(th) panel; 42% G2/M by DAPI, and only 13% ofthe cells BrdU+).

These results are completely consistent with the fact that at the lowerdose of SCCP ID No. 6037, insufficient PLK1 inhibition results in thecells entering mitosis, dividing, and then arresting in G1 where PLK3inhibition prevents entry into S phase. At the higher dose, PLK1 isinhibited as the cells exit from S phase and then accumulate in G2/M.The putative PLK3-dependent G1 arrest was only revealed at a lower doseat which PLK1 inhibition is not predominating. Thus, the disclosed PLKselective inhibitors can provide a route for study and understanding ofcell cycles, and the disclosed PLK selective inhibitors can function asa chemical probe useful in mechanism of action studies.

Disclosed small molecule inhibitors can also encourage apoptosis. Forinstance, cell cycle analysis and a caspase apoptosis assay haveindicated high levels of apoptosis for SCCP ID No. 6037 and SCCP ID No.5915 in contrast to virtually none for the negative control SCCP ID No.5914, suggestive of target engagement for the small molecule inhibitors.

The small molecule inhibitors can be developed and synthesized accordingto methods as are generally known in the art. For instance, in oneembodiment, an iterative combinatorial method can be utilized asdescribed in U.S. Pat. No. 9,175,357 to McInnes, et al., which isincorporated herein by reference. Overall, this combinatorial approachis based upon a known peptide inhibitor and allows both regions of theinhibitor molecule (i.e., the N-cap and the C-cap) to be optimizedindependently to maximize the affinity and drug-likeness of eachcomponent. FIG. 4 provides a general scheme for synthesis according tothis particular embodiment. Peptide inhibitors that can be utilized asthe basis for development of the small molecule inhibitors can includeany peptide inhibitor capable of selectively inhibiting PLK. Forinstance, the basis peptide inhibitor can include both native peptidesand variants thereof.

Synthesis of the inhibitors can be carried out according to any suitablechemical process. By way of example, and without limitation, FIG. 5provides three possible schemes for inhibitors: 1) FIG. 5 ; 2) scheme Apresents a methodology for incorporating aryl groups onto the linker;and 3) scheme B presents one methodology for derivatization of theinhibitor at the R₄ and R₅ positions of the above described structures(R₁, R₂, on scheme B). This scheme can also be utilized to modulate theR₂ position (e.g., an R₂ carboxylate in the above described structures)by addition of electron withdrawing groups and replacement with, e.g.,phosphate isosteres.

A benefit of the iterative combinatorial formation approach is themodularity in identification of desired moieties for each subsite. Thedesired fragments can be combinatorially ligated so that essentialcompound features will not be compromised during the linking process.The individual fragments can be optimized for potency and drug-likeproperties and then linked to improve pharmacodynamic andpharmacokinetic properties. This lead optimization stage can be informedby cellular assays and detailed mechanistic studies of anti-tumoreffects of the inhibitors. The modularity and combinatorial aspects ofthis method can allow for the moieties to be exchanged and more narrowlytarget the characteristics of the physicochemical characteristics, aswell as minimize metabolic or toxicophore liabilities, with particularlypreferred characteristics depending primarily on the application of theinhibitor (e.g., research, in vivo).

According to one embodiment, following identification and optimizationof the end groups, a bridging strategy between the benzamido moiety andan aminobenzoic acid moiety can be selected. For instance, the bridgebetween the aryl groups can include an amide, ether, thioether, amine,or carbon-carbon linkages.

The fragments can be subdivided into chemotypes appropriate for dockinginto the particular PBD sub-sites and prioritized using pharmacophorefeatures to select fragments with desired functionality. High-throughputdocking of virtual libraries into the PBD binding site can be performedduring the synthesis process. High-throughput docking (HTD) can then beused in refinement of the fragments with calculations including moreaccurate scoring functions, and interactions filters to limit fragmentsto those containing the geometrically appropriate functionality. HTDprograms such as, without limitation, LIDAEUS, LigandFit, Accelrys®, andGlide (Schrödinger®), can incorporate the desired enhancements and canbe used to dock virtual fragment libraries into each site. For example,when calculations are parallelized, 100,000 fragments can be screenedbetween 5 and 50 hours depending on the parameterization and number ofCPU's employed. Due to inherent inaccuracies with docking, it can beexpedient to use different implementations to minimize errors, biases,or incorrect parameters of a single synthesis process. A balance ofelectrostatic, van der Waals, and H-bonding interactions between eachfragment and the binding groove can be used to form the inhibitor havingthe desired characteristics.

After development of the inhibitor fragments from identified chemotypes,these can be combined in order to substitute all peptidic determinantsand form the non-peptidic small molecule inhibitors. Optimization of thefragments can be facilitated using 3-D structures generated throughcrystallography and flexible molecular docking (using, e.g., Cdocker,Accelrys®, etc.) to predict favorable interactions of modified compoundsand improve complementarity.

The present disclosure may be better understood with reference to theExamples, set forth below.

Example 1

Materials and Methods

Fluorescence Polarization Assay

Materials were dissolved in DMSO (10 mM) and diluted from 10 nM to 600μM (maximum of 6% DMSO tolerance in the assay). PLK1 PBD (367-603) andPLK3 PBD (335-646) proteins were obtained from BPS Biosciences Inc. (SanDiego, Calif.); 17 ng PLK1 and 156 ng PLK3 were used per reaction. Thefluorescein-tracer phospho-peptides (MAGPMQS[pT]PLNGAKK (SEQ ID NO: 1)for PLK1, and GPLATS[pT]PKNG (SEQ ID NO: 2) for PLK3) were used at afinal concentration of 10 nM. Incubation was carried out at roomtemperature for 45 minutes. Fluorescence was measured using a DTX 880plate reader and Multimode Analysis software (Beckman Coulter, Brea,Calif.). The polarization values in millipolarization (mP) units weremeasured at an excitation wavelength of 488 nm and an emissionwavelength of 535 nm. Each data point was performed in triplicate forevery experiment, and experiments were performed at least three times.An IC₅₀ value for each compound was calculated from linear regressionanalysis of the plots.

PLK1 Kinase Inhibition Assay

The CycLex® Polo-like Kinase 1 Assay/Inhibitor Screening Kit was used tomeasure catalytic inhibition (MBL Life Science, Nagano, Japan). ThisELISA assay measures the catalytic activity of full length PLK1 for adefined substrate, which is detected by an anti-phospho-threonineantibody (PPT-07) and peroxidase coupled secondary antibody. Platespre-coated with a Threonine-containing substrate were incubated withPLK1, kinase buffer containing 7.5 μM ATP (modification from recommendedconcentration), in the absence or presence of increasing concentrationof inhibitor. After incubation, the phosphorylated substrate resultingfrom PLK1 kinase activity was detected using the PPT-07 antibody andhorseradish peroxidase conjugated anti-rabbit IgG antibody. Peroxidasecatalyzes the conversion of the colorless solution to yellow, which wasquantified using a DTX 880 plate reader. Absorbance measurements (450nm) were plotted to calculate activity in each sample, and % inhibitionwas calculated in the wells relative to activity in the absence ofinhibitor.

Cell Culture

HeLa cervical cancer cells were obtained from ATCC (Manassas, Va.).Histone H2B GFP-labeled HeLa cells (HeLa-H2B-GFP) were kindly providedby Dr. Geoffrey Wahl (Gene Expression Laboratory, Salk Institute), andwere confirmed as >95% GFP positive by FACS (data not shown) but werenot otherwise authenticated. Cells were maintained in DMEM (Invitrogen™,Carlsebad, Calif.) supplemented with 10% FBS or Corning© Nu-Serum™ (BD™Biosciences, Franklin Lakes, N.J.) and 1% penicillin/streptomycin(Invitrogen™) in a humidified incubator and 5% CO₂ at 37° C.

PC3 prostate cancer cells were maintained in DMEM (Invitrogen™,Carlsebad, Calif.) supplemented with 10% FBS or Corning® Nu-Serum™ (BD™Biosciences, Franklin Lakes, N.J.) and 1% penicillin/streptomycin(Invitrogen™) in a humidified incubator and 5% C02 at 37° C.

A-549 lung cancer cells were maintained in Hams F-12 1× with glutamine(Corning® Cellgro, Manassas, Va.), and supplemented with 10% FBS orCorning® Nu-Serum™ (BD™ Biosciences, Franklin Lakes, N.J.) and 1%penicillin/streptomycin (Invitrogen™) in a humidified incubator and 5%C02 at 37° C.

HCT-116 (p53+/+ and −/−) colon cancer cells were maintained DMEM(Invitrogen™, Carlsebad, Calif.) supplemented with 10% FBS or Nu-serum(BD™ Biosciences, Franklin Lakes, N.J.) and 1% penicillin/streptomycin(Invitrogen™) in a humidified incubator and 5% C02 at 37° C.

Retinal Pigment Epithelial (RPE) cells were maintained in 1:1 DMEM/HamsF-12 (Invitrogen™, Carlsebad, Calif.; Corning® Cellgro, Manassas, Va.),and supplemented with 10% FBS or Corning® Nu-Serum™ (BD™ Biosciences,Franklin Lakes, N.J.) and 1% penicillin/streptomycin (Invitrogen™) in ahumidified incubator and 5% C02 at 37° C.

Cell Viability Assay

Exponentially growing cells were plated in 96-well dishes. Dose responsecurves were used to treat cells for 72 hours with inhibitors. Followingthe three-day treatment, cell viability was measured using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)colorimetric assay. Cells were incubated for 3 hours with MTT allowingviable cells to metabolize the tetrazolium dye to a purple coloredsolution. Absorbance measurements (595 nm) were quantified using a DTX880 plate reader and IC₅₀ values were generated for FLIPs.

Analysis of Cell Cycle Progression by FACS

Serum-Starved Synchronization

Cells were synchronized in G1 by serum starvation. Briefly,exponentially growing cells were plated overnight. Media (DMEM, 10% FBS,1% Pen. Strep.) was then removed and replaced with media containing noFBS. Cells were starved for 72 hours then treated for 24 hours withinhibitor. Following treatment, cells were harvested and processed forendpoint experimental analysis.

BrdU Labeling/FACS

Ethanol fixative was removed by centrifugation and the cells were washedwith 1 mL ice-cold PBS/1% BSA. Cells were denatured by resuspending in0.2 mg/mL pepsin in 2N HCl and incubated in 37° C. water bath for 15minutes. Hydrolysis was then terminated by adding 1 M Invitrogen™ Novex®Tris-Glycine. Cells were then washed with PBS/1% BSA then the primaryanti-BrdU antibody (1:100 dilution in TBFP; 0.5% Tween-20, 1% BSA, 1%FBS, PBS) was added and cells were incubated for 25 minutes in the dark,at room temperature. Cells were subsequently washed with PBS/1% BSA thenallowed to incubate for 25 minutes in the secondary fluorescent antibody(1:200 dilution Alexa Fluor® 488 F(ab′)₂ fragment of goat anti-mouseIgG). Cells were washed once more with PBS/1% BSA and re-suspended inDAPI for 30 minutes before being analyzed by flow cytometry.

Example 2

PBD-Inhibitors Bind Potently to the PLK1-PBD and are Selective

Substructure searching for 4-alkybenzamide derivatives in commerciallibraries and subsequent testing identified one lead molecule inhibitor,SCCP ID No: 5881. From this lead, the structure activity relationship(SAR) of more than forty small molecules was analyzed. The total ofthese compounds can be found in FIG. 1 . Specifically, compound bindingto PLK1 (discussed first) and PLK3 (discussed second) in vitro wasanalyzed. ATP inhibition was also measured and compound activityanalyzed in cancer cells.

Structure Activity Relationship of C-Capping Group on Benzamide SmallMolecules

SCCP ID No: 5880 (butyl-benzamide) did not bind to the PBD of PLK1,however SCCP ID No: 5905 (pentyl-benzamide) bound to the PLK1 PBD withweak affinity (IC₅₀=223.6±4.88 μM). When the alkyl tail length wasincreased from pentyl to hexyl PLK1 binding increased. For instance, forSCCP ID No: 5881, binding increased about 12-fold (IC₅₀=18.4±5.3 μM).SCCP ID No: 5881 also bonded to PLK3 (IC₅₀=18.04±5.18 μM) but remained5-fold selective for PLK1. An increase in binding for PLK1 was seen byfurther lengthening (octyl) the alkyl tail (e.g., SCCP ID No: 5912)(IC₅₀=11.27±2.7 μM). Selectivity was also marginally improved (PLK3IC₅₀=15.48±0.01 μM), relative to SCCP ID No: 5881. Further lengtheningof the alkyl tail was carried out to determine if PLK1 binding would befurther increased. Increasing the carbon chain to 12 carbons resulted inthe generation of the least selective small molecule SCCP ID No: 6037(PLK1 IC₅₀=5.97±0.38; PLK3 IC₅₀=1.99±0.69; Fold=1.6) as shown in Table2, below.

TABLE 2 PLK1 Kinase SCCP PLK1 IC₅₀ PLK3 IC₅₀ Selectivity Inhibitory Act.ID No. (μM) (μM) Fold Index IC₅₀ (μM) 5880 >600 ND ND ND 5905 223.6 ±4.88 ND ND ND 5881 18.4 ± 5.3 18.04 ± 5.18 5 ND 5912 11.27 ± 2.7  15.48± 0.01 6.9 42.36 6037  5.97 ± 0.38  1.99 ± 0.69 1.6 ND

Several of the inhibitors were examined upon variation on the C-terminalfragment. Results are provided in Table 3, below. When the position ofthe carboxylic acid was moved from ortho to meta (from the alanine), aloss of PLK1 binding affinity was observed (SCCP ID No: 5903IC₅₀=129.8±3.6 μM) with minimal selectivity (PLK3 IC₅₀=43.62±1.69 μM).No binding was observed with a sulfonamide phospho-mimic (SCCP ID No:5908), or when using a phosphate (SCCP ID No: 5940) or hydroxyl (SCCP IDNo: 5943) group. Interestingly, when a methyl group was positioned metato the carboxylic acid (SCCP ID No: 5924), PLK3 PBD binding was observed(IC₅₀=24.5 μM), but no binding to PLK1.

TABLE 3 SCCP PLK1 IC₅₀ PLK3 IC₅₀ Selectivity ID No: (μM) (μM) Fold Index5881  18.4 ± 5.3 18.04 ± 5.18 5 5903 129.8 ± 3.6 43.62 ± 1.69 1.65908 >600 ND ND 5924 >600 24.5 ND 5940 >600 ND ND 5943 >600 ND ND

Several derivatives of the octyl-containing SCCP ID No: 5912 weresynthesized and an analysis of the SAR of substituents on the C-terminalreplacement was analyzed. As shown in Table 4, below as expected due tothe putative contribution of the negatively charged carboxylate group,PLK1 binding was lost when the carboxylic acid was converted to a ethylester (SCCP ID No: 5914; IC₅₀=>600 μM). This compound was then used as anegative control for cellular analysis.

TABLE 4 PLK1 Kinase SCCP PLK1 IC₅₀ PLK3 IC₅₀ Selectivity Inhibitory Act.ID No: (μM) (μM) Fold Index IC₅₀ (μM) 5912 11.27 ± 2.7  15.48 ± .01  6.9 42.36 5914 >600 ND ND ND 5932 11.1 ± 1.1 13.24 ± 0.46  6 48.9 5915 2.16 ± 0.01 7.68 ± 2.41 17.8 24.3 5937 3.99 ± 2.3 7.42 ± 1.89 9.3 ND

When SCCP ID No: 5932 (Table 4) was synthesized containing a fluorineortho to the carboxylic acid, PLK1 PBD binding affinity did not improve(relative to SCCP ID No: 5912, Table 4), nor was PLK1 selectivityincreased. When fluorine was added at both the ortho and para positions(SCCP ID No: 5938, Table 5), PLK1 PBD binding decreased (IC₅₀=30.6±3.2μM) as did PLK3 binding (IC₅₀=39.7 μM).

TABLE 5 SCCP PLK1 IC₅₀ PLK3 IC₅₀ Selectivity ID No: (μM) (μM) Fold Index5935  5.89 ± 1.25 13.6 ± 6.3  11.5 5938 30.6 ± 3.2 39.7 6.5 5939 6.05 ±1.9 9.96 ± 1.52 8.2

Improved PLK1 binding (IC₅₀=2.16±0.01 μM) and selectivity (PLK3IC₅₀=7.68±2.41 μM, Fold Selectivity=18) was observed upon addition of amethyl group meta to the carboxylic acid (SCCP ID No: 5915, Table 4). Itis important to note that the only difference between this compound andSCCP ID No: 5924 (Table 3) is the length of the alkyl tail. Increasingthe length from hexyl (SCCP ID No: 5924, Table 3) to octyl (SCCP ID No:5915, Table 4) dramatically improved PLK1 binding and produced the mostselective compound in this series (Table 4). When the methyl group waspositioned para to the carboxylic acid (SCCP ID No: 5937, Table 4), PLK1selectivity decreased 2-fold relative to SCCP ID No: 5915 (compareselectivity fold 18 with 9.3).

A compound containing a methoxy group positioned meta to the carboxylicacid (SCCP ID No: 5935, Table 5) was 11.5-fold selective for PLK1,binding with an IC₅₀=5.89±1.25 μM.

SCCP ID No: 5939 (Table 5) contains a nitro group ortho to thecarboxylic acid. It is 8-fold selective for PLK1, binding to the PLK1PBD with an IC₅₀=6.05±1.9 μM (PLK3 PBD IC₅₀=9.96±1.52 μM).

Small molecule inhibitors containing an alkyl benzamine flexiblestructure were also synthesized to explore the SAR of compounds withless rigidity. Results are shown in Table 6, below. For instance, SCCPID No: 5953 was synthesized with a methylamino instead of the amidelinkage found in SCCP ID No: 5912 (Table 4). Binding to PLK1 wasslightly improved (SCCP ID No: 5953 IC₅₀=9.57±2.03), relative to SCCP IDNo: 5912. Next, SCCP ID No: 5961 contains the methylamino linker and amethyl group meta to the carboxylic acid—the same substitution at the R₄position as SCCP ID No: 5915 (Table 4). Binding of SCCP ID No: 5961 tothe PLK1 PBD was undetectable, but it did have weak affinity for thePLK3 PBD (IC₅₀=156.7 μM). This suggests an important conformational rolefor compound binding. Positioning of a methyl group para to thecarboxylic acid (e.g., SCCP ID No: 5971), resulted in improved PLK1binding (IC₅₀=21.69±2.8 μM) relative to SCCP ID No: 5961. Thisrepresents more than 27-fold increase in PLK1 binding relative to SCCPID No: 5961, however there was a 2-fold loss in binding relative to SCCPID No: 5953. This further suggests that the conformation of the compoundwithin the binding pocket is important for the methyl group substituent.

TABLE 6 SCCP PLK1 IC₅₀ PLK3 IC₅₀ Selectivity ID No: (μM) (μM) Fold Index5953 9.57 ± 2.03 ND ND 5961 >600 156.7 ND 5971 21.69 ± 2.8  ND ND

The ability of the most promising compounds to inhibit the catalyticactivity of PLK1 was investigated using an in vitro ELISA-based PLK1kinase assay. The PBD-based PLK1 inhibitors (SCCP ID Nos: 5912, 5932,and 5915) inhibited the catalytic activity of the protein (IC₅₀=42.3 μM,48.9 μM, and 24.3 μM, respectively) (Table 4). The results from thisassay were an important confirmation of compound binding to PLK1 in anorthogonal assay.

The most promising inhibitors of FIG. 1 were tested in multiple cancercell lines, including HeLa cervical cancer cells, PTEN deficientprostate cancer (PC3) cells, and KRAS mutant lung cancer (A-549) cells.Results are shown in Table 7, below. Each of these cancer cells lineswere sensitive to the lead PBD-inhibitors in a cell viability assay,with the A-549 lung cancer cells being the most sensitive overall. Thisis consistent with literature stating a synthetic lethal interactionexists with PLK1 mutant RAS cancer cells. A synthetic lethal interactionhas also been demonstrated between PLK1- and PTEN-deficient prostatecancer cells. A differential sensitivity was also observed depending onp53 status. Specifically, p53 null HCT 116 colon cancer cells were moresensitive to the PBD-inhibitors relative to p53 proficient HCT 116cells.

TABLE 7 HCT116 HCT116 SCCP HeLa PCC-3 A-549 (p53−/−) (p53+/+) ID No:(μM) (μM) (μM) (μM) (μM) 5912 15.2 ± 1.4 15.1 ± 1.7 2.8 ± 0.1  8.0 ± 1.714.8 ± 6.4 5914 >100 >100 ND ND ND 5915 15.5 ± 1.7 16.3 ± 2.1 5.1 ± 1.510.0 ± 1.0 17.4 ± 5.6 5932 12.7 ± 2.3 11.6 ± 1.2 2.9 ± 0.5 10.0 ± 2.412.4 ± 2.7 5937 17.9 ± 2.1 18.8 ± 1.7 3.5 ± 1.2 11.4 ± 1.6 24.2 ± 7.15945 20.1 ± 3.1 27.9 ± 2.6 13.3 ± 0.8  24.9 ± 2.0 ND 5946 33.6 ± 2.634.2 ± 2.5 ND ND ND 6037 14.0 ± 3.5  9.4 ± 0.8 ND ND ND

It has previously been demonstrated that a single point mutation withinthe catalytic domain of PLK1 confers resistance to ATP-based inhibitorswhile retaining full catalytic activity. The Retinal Pigment Epithelial(RPE) cells generated by this group were obtained and the resistantphenotype was confirmed by cell viability assay (FIG. 6 ). As shown, theresults confirm that WT RPE cells are sensitive to BI-2536 (IC₅₀=21.2nM) while mutant RPE cells are dramatically resistant to BI-2536 (>2.5μM). Non-peptidic PBD inhibitors were found to be able to circumvent theresistance observed by ATP-based inhibitors using this RPE cell line.Cells containing wild type and mutant (C67V) PLK1 were sensitive to leadPBD-inhibitors (SCCP ID No: 5912 (FIG. 7 ) and SCCP ID No: 5932 (FIG. 8)) while C67V mutant cells were resistant to BI-2536—an ATP-competitivePLK1 inhibitor (FIG. 6 ).

The PLK1 PBD-inhibitors were analyzed further by cell cycle analysis inPC3 cells (FIG. 9 ). At a concentration of 5 μM, SCCP ID No: 5912 showsmoderate (13% increase relative to control) increase in G2/M population,suggesting PLK1 is being inhibited (FIG. 10 ). This result is modestwhen compared to those obtained using an ATP-competitive inhibitor(BI-2536), which shows nearly 80% of cells accumulate in G2/M. A higherconcentration of SCCP ID No: 5912 (15 μM) results in a phenotype thatsuggests other targets are being engaged (FIG. 9 ). SCCP ID No: 5912shows S phase delay (FIG. 9 and FIG. 10 ), implicating PLK3 inhibitionat 15 μM; whereas SCCP ID Nos: 5915 and 5935 show G1 delay or arrest at15 μM, implicating PLK2 or PLK3 inhibition. To quantify the percentageof cells actively proliferating (synthesizing their DNA) or arrestedBromodeoxyuridine (BrdU) pulse labeling was carried out and analyzed byflow cytometry. Results showed that 28.3% of cells were replicatingtheir DNA when treated with 5 μM SCCP ID No: 5915; however, cells arearrested in G1 (BrdU+=3.6%) following treatment with 15 μM SCCP ID No:5915.

After treating PC3 cells with SCCP ID No: 6037—the least selectiveinhibitor (Fold Selectivity=1.6)—G1 accumulation was observed at boththe lower and higher dose (FIG. 11 ). Upon low dose (5 μM) treatment,BrdU labeling showed that 75.7% of the cells were replicating their DNA.This suggests that the lower dose observations result from either adelay of cells exiting G1 or delayed progression through S phase, asopposed to an S-phase arrest. Like SCCP ID No: 5915, a G1 arrest wasobserved following the higher dose (15 μM) treatment (BrdU+=1.9%). Thissupports the FP data showing low PLK1 selectivity for SCCP ID No: 6037.

Additional cell viability data for the inhibitors is provided in FIG. 12-FIG. 14 . For instance, FIG. 12 presents cytotoxicity, cell viability,and apoptosis data for cells treated with inhibitor SCCP ID No: 6037.The average from various essays included a cell viability of 15.6±3.4μM, a cytotoxicity of 14.8±3.6 μM, and apoptosis of ±5.6 μM. The data ofFIG. 13 relates to SCCP ID No: 5914 and the data of FIG. 14 relates toSCCP ID No: 5915.

It will be appreciated that the foregoing examples, given for purposesof illustration, are not to be construed as limiting the scope of thisdisclosure. Although only a few exemplary embodiments have beendescribed in detail above, those skilled in the art will readilyappreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this disclosure. Accordingly, all such modifications areintended to be included within the scope of this disclosure, which isdefined in the following claims and all equivalents thereto. Further, itis recognized that many embodiments may be conceived that do not achieveall of the advantages of some embodiments, yet the absence of aparticular advantage shall not be construed to necessarily mean thatsuch an embodiment is outside the scope of the present disclosure.

What is claimed is:
 1. A method for inhibiting Polo-Like Kinase (PLK)proteins, the method comprising: providing a small molecule PLKinhibitor to a medium containing one or more PLK proteins, wherein thesmall molecule PLK inhibitor has the general structure:

wherein R1 is an -S-alkyl; and R₄ is an alkyl.
 2. The method of claim 1,wherein the medium comprises a solution.
 3. The method of claim 1,wherein the medium comprises one or more cells.
 4. The method of claim3, wherein the one or more cells comprise HeLa cervical cancer cells,PTEN-deficient prostate cancer PC3 cells, KRAS mutant lung cancer A-549cells, or a combination thereof.
 5. The method of claim 1, wherein themedium comprises one or more cells and the inhibitor induces apoptosisin a least a portion of the one or more cells.
 6. The method of claim 1,wherein the one or more Polo-like Kinase proteins comprise PLK3.
 7. Themethod of claim 1, wherein the one or more Polo-like Kinase proteinscomprise PLK1.
 8. The method of claim 1, wherein the small molecule PLKinhibitor has an atomic mass of about 1000 Daltons or less.
 9. Themethod of claim 1, wherein the small molecule PLK inhibitor has anatomic mass of about 500 Daltons or less.
 10. The method of claim 1,wherein the small molecule PLK inhibitor has a polar surface area ofabout 200 Å or less.
 11. The method of claim 1, wherein the smallmolecule PLK inhibitor has a polar surface area of about 100 Å or less.12. The method of claim 1, wherein R4 is a methyl.